Tubulin, the major component of microtubules, undergoes a posttranslational modification that is unique to this protein. The carboxy terminal amino acid on just one of the highly homologous subunits of the tubulin heterodimer is hydrolyzed and replaced by a cycle of two specific enzymes. Tubulin tyrosine carboxypeptidase (TTCP) removes the genetically encoded C-terminal tyrosine from polymerized tubulin, exposing a glutamic acid residue. Tubulin tyrosine ligase (TTL) catalyzes peptide bond formation between this glutamic acid and tyrosine, and this enzyme acts preferentially on tubulin in its depolymerized state. TTL has been isolated and cloned, but nothing is known about the sequence and structure of TTCP. This post-translational modification cycle is critical for neuronal network organization and its disruption can also affect tumorigenesis. TTL is downregulated in some aggressive cancers;thus, molecules that promote tubulin tyrosination - perhaps TTCP inhibitors - may be useful chemotherapeutic agents for these cancers, which currently have poor prognosis. The goal of this research project is to develop novel tools for in depth study of tubulin tyrosination-detyrosination using chemical biology. We intend to develop a new bioorthogonal system for labeling tyrosinated tubulin that involves enzymatic attachment of the reactive amino acid 3- formyl tyrosine to the C-terminus of a-tubulin. Novel fluorescent probes with a complementary reactive group will be synthesized for coupling to the modified protein. A unique feature of these probes is that they are weakly fluorescent until they react with the modified amino acid, therefore yielding negligible background fluorescence from unreacted fluorophore Both the enzymatic reaction and the chemical coupling are designed to take place in live cells;thus, tubulin tyrosination in live cells may be observed both temporarily and spatially. By attaching appropriate probes directly to a defined site on polymerized tubulin, superior ultra high resolution images of microtubules using sub-diffraction microscopy techniques should be attainable. TTL will also be used to identify the elusive TTCP by photoaffinity labeling. Eventual isolation of purified TTCP will allow for the development of screening assays to find inhibitors of this enzyme, which may become anti-cancer agents to combat aggressive tumors. PUBLIC HEALTH RELEVANCE: Microtubules are part of the cytoskeleton in all human cells. These structures grow and shrink in size in response to the needs of the cell. One enzyme involved in this process is tubulin tyrosine ligase. Many cancer cells are deficient in this enzyme, and it is possible that loss of this enzyme's activity is associated with particularly aggressive cancer. This research project will examine the activity of tubulin tyrosine ligase using fluorescent molecules. This enzyme will also be used as a tool to obtain high resolution pictures of microtubules in cells.